DESCRIPTION: This is a competitive renewal of a program which has been funded by NIH for the past twelve years. The long term objective of the PI's work is to determine the neural and endocrine mechanisms that control sexual differentiation of brain and behavior. The PI has employed zebra finches because 1) only the males sing a courtship song, 2) the brain regions controlling song are much larger in males than in females, and 3) the development of song and the masculine pattern of neural development is triggered early in development by estrogen. The PI has made excellent progress during the previous funding period. Even though the funds were cut from five to three years, the PI was able to accomplish much of the work outlined in the proposal. The first aim was to use intracerebral estrogen implants into female zebra finches to localize the neural sites of estrogen action. The PI found the HVC to be an effective site for masculinization of the female song system by estrogen. Androgens were found to be ineffective in masculinizing the female zebra finches. The second aim was to search for sex differences in estrogen synthesis; few were found. The third aim was to use tissue cultures of brain cells from hatchling and adult zebra finches to examine the capacity of different cell types to synthesize estrogen, to express estrogen receptors, and to synthesize other sex steroids. The PI has now set up tissue culture in his own laboratory and has found that aromatase is expressed in both neurons and glia. The fourth aim was to treat zebra finch embryos with hormones, blocking agents, and aromatase inhibitors to study the impact of these manipulations on sexual differentiation of the song system. Fadrozole was used to inhibit estrogen synthesis by blocking aromatase, and thus the metabolic conversion of testosterone to estradiol. The results indicated that inhibition of estrogen synthesis has little effect on masculine development of song. The PI also demonstrated that the brain is the main source of circulating estrogens in male zebra finches. In addition, the PI s laboratory has cloned the zebra finch estrogen receptor and aromatase, so that these molecular tools are now available for use in future studies. In sum, the PI has made superb progress during the past three years. Data emerging from the PI's laboratory suggests that the standard model, (ie. that estrogens and aromatizable androgens are responsible for masculinization) may be incorrect. He has conflicting evidence suggesting that androgens do not masculinize females, and that inhibition of estrogen synthesis fails to prevent masculinization in males. In order to resolve these conflicts, the PI has put forth an attractive, resourceful and complex hypothesis to test whether estrogen is a masculinizing or a feminizing hormone in zebra finches. The specific aims are 1) determine the role of estrogen in ovo, on gonadal development and sexual differentiation of the brain using steroid synthesis inhibitors and receptor blockers. The second aim is to investigate the role of the ovary in sexual differentiation of the brain by transplanting ovarian tissue into hatchling and embryonic males, and by neonatal ovariectomy in females. The third aim is to establish the developmental characteristics of estrogen and androgen receptor expression. The fourth aim is to measure, using in situ hybridization, the effects of estrogen on the expression of three types of genes (AR s, two immediate early genes, and neurotrophin receptors) which may be related to the dimorphic development of bird song. The fifth aim is to measure the effects of androgens and estrogens on neurons and non- neuronal cells in primary telencephalic cultures.